Investigation of ligand interactions with human RXRα by hydrogen/deuterium exchange and mass spectrometry

Several different agonists of the retinoic X receptor alpha (hRXRalpha) were examined for their effects on the amide H/D exchange kinetics of the homodimeric protein using mass spectrometry. Some agonists, LG 100268, SR11246, and DHA, bind such that slower deuterium exchange-in occurs compared with 9-cis-retinoic acid (9-cis-RA), whereas others, fenretinide and methoprenic acid, result in poorer protection during binding and hence faster exchange-in. Protection against H/D exchange by different agonists and the inhibition of H/D exchange kinetics relative to 9-cis-RA varies markedly in different regions of the protein. Agonists LG 100268, SR11246, and DHA generally inhibit faster exchange processes in the ligand binding regions of hRXRalpha than does the native ligand 9-cis-RA. In at least half of these regions, the level of protection by 9-cis-RA lags behind the agonists even after 60 min. Methoprenic acid did not significantly protect hRXRalpha against amide hydrogen exchange. An efficient method is described for comparing the effects of different agonists on the protein structure of the agonist-RXRalpha complex.     

Protein-metal interactions of calmodulin and α-synuclein monitored by selective noncovalent adduct protein probing mass spectrometry

The metal binding properties of proteins are biologically significant, particularly in relationship to the molecular origins of disease and the discovery of therapeutic pharmaceutical treatments. Herein, we demonstrate that selective noncovalent adduct protein probing mass spectrometry (SNAPP-MS) is a sensitive technique to investigate the structural effects of protein-metal interactions. We utilize specific, noncovalent interactions between 18-crown-6 ether (18C6) and lysine to probe protein structure in the presence and absence of metal ions. Application of SNAPP-MS to the calmodulin-Ca²⁺ system demonstrates that changes in protein structure are reflected in a substantial change in the number and intensity of 18C6s, which bind to the protein as observed by MS. In this manner, SNAPP is demonstrated to be a sensitive technique for monitoring ligand-induced conformational rearrangements in proteins. In addition, SNAPP is well-suited to examine the properties of natively unfolded proteins, where structural changes are more difficult to detect by other methods. For example, α-synuclein is a protein associated in the pathology of Parkinson’s disease, which is known to aggregate more rapidly in the presence of Al³⁺ and Cu²⁺. The 18C6 SNAPP distributions for α-synuclein change dramatically in the presence of 3 μM Al³⁺, revealing that Al³⁺ binding causes a significant change in the conformational dynamics of the monomeric form of this disordered protein. In contrast, binding of Cu²⁺ does not induce a significant shift in 18C6 binding, suggesting that noteworthy structural reorganizations at the monomeric level are minimal. These results are consistent with the idea that the metal-induced aggregation caused by Al³⁺ and Cu²⁺ proceed by independent pathways.     

Analysis of chemical profiles of insect adhesion secretions by gas chromatography–mass spectrometry

This article reports on the chemical analysis of molecular profiles of tarsal secretions of the desert locust Schistocerca gregaria (Forsskål, 1775) by gas chromatography hyphenated with quadrupol mass spectrometry (GC–MS) as well as ¹H-nuclear magnetic resonance (¹H NMR) spectroscopy. Special focus of this study was to elaborate on sampling methods which enable selective microscale extraction of insect secretions in a spatially controlled manner, in particular tarsal adhesive secretions and secretions located on cuticle surfaces at the tibia. Various solvent sampling procedures and contact solid-phase microextraction (SPME) methods were compared in terms of comprehensiveness and extraction efficiencies as measured by signal intensities in GC–MS. Solvent sampling with water as extraction solvent gave access to the elucidation of chemical profiles of polar compound classes such as amino acids and carbohydrates, but is extremely tedious. Contact SPME on the other hand can be regarded as a simplified and more elegant alternative, in particular for the lipophilic compound fraction. Many proteinogenic amino acids and ornithine as well as carbohydrate monomers arabinose, xylose, glucose, and galactose were detected in tarsal secretions after acid hydrolysis of aqueous extracts. Qualitatively similar but quantitatively significantly different molecular profiles were found for the lipid fraction which contained mainly n-alkanes and internally branched monomethyl-, dimethyl-, and trimethyl-alkanes in the C23–C49 range as well as long chain fatty acids and aldehydes. Especially, hydrocarbons with >C40 carbon numbers have previously been rarely reported for insect secretions. The results suggest that the investigated insect secretions are complex emulsions which allow the attachment of tarsi on various otherwise incompatible materials of smooth and rough surfaces. The solid consistence of the established alkanes at ambient temperatures might contribute to a semi-solid consistence of the adhesive, amalgamating partly opposing functions such as slip resistance, tarsal release, desiccation resistance, and mechanical compliance. The methods developed can be extended to other similar applications of studying compositions of insect secretions of other species.     

Recent developments in protein–ligand affinity mass spectrometry

This review provides an overview of direct and indirect technologies to screen protein–ligand interactions with mass spectrometry. These technologies have as a key feature the selection or affinity purification of ligands in mixtures prior to detection. Specific fields of interest for these technologies are metabolic profiling of bioactive metabolites, natural extract screening, and the screening of libraries for bioactives, such as parallel synthesis libraries and small combichem libraries. The review addresses the principles of each of the methods discussed, with a focus on developments in recent years, and the applicability of the methods to lead generation and development in drug discovery.     

Identification of polyethanolamines by chromatography–mass spectrometry

Presumable structures of polyethanolamines, synthesized by the catalytic β-hydroxyethylation of ammonia with an excess of ethylene oxide are determined by gas chromatography–electron ionization mass spectrometry and tandem mass spectrometry qualitatively, following fragmentation ways and also taking into account retention data. The preferable paths of consecutive reactions of the synthesis of polyethanolamines in a homologous series of isomers are found.     

Characterization of impurities in tobramycin by liquid chromatography–mass spectrometry

The characterization of unknown impurities present in tobramycin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. A reversed-phase (RP)-LC method using a volatile mobile phase containing a perfluorinated ion-pair reagent was developed and coupled with an ion trap mass spectrometer. The structures of the unknown impurities were deduced by comparison of their fragmentation patterns with those of the available reference substances obtained by LC–MSⁿ experiments.     

Optimization of antibiotic analysis in water by solid-phase extraction and high performance liquid chromatography–mass spectrometry/mass spectrometry

This paper describes the development of an optimized method based on solid-phase extraction (SPE) followed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) for the simultaneous analysis of ten antibiotic compounds including tetracyclines, sulfonamides, macrolides and quinolones. LC–MS/MS sensitivity has been optimized by alterations to both LC and MS operations. Of the two high resolution columns tested, Waters Symmetry C₁₈ endcapped and Agilent Zorbax Bonus-RP, the latter was found to show better performance in producing sharp peaks and clear separation for most of the target compounds. Optimization of the MS fragmentation collision and cone energy enhanced the peak areas of the target analytes. The recovery of the target compounds from water samples was most efficient on Waters Oasis HLB SPE cartridge, while methanol was shown to be the most suitable solvent for desorbing the compounds from SPE. In addition, acidification of samples prior to SPE was shown to enhance the recovery of the compounds. To ensure a satisfactory recovery, the flow rate through SPE should be maintained at ≤10 mL min⁻¹. The method was successfully applied to the analysis of antibiotics from environmental water samples, with concentrations being <LOD in tap water, between <LOD to 28 ng L⁻¹ in river water and between <LOD to 230 ng L⁻¹ in sewage effluent.     

Determination of organophosphate flame retardants in sediments by microwave-assisted extraction and gas chromatography–mass spectrometry with electron impact and chemical ionization

An efficient microwave-assisted extraction (MAE) procedure coupled to gas chromatography–mass spectrometry (GC–MS) with electron impact (EI) and chemical ionization (CI) has been developed to determine five organophosphate flame retardants (OPFRs) in marine and river sediments. The effects of various operating parameters on the quantitative extraction of the OPFRs through MAE were systematically investigated. Selected OPFRs were extracted from the sediments through MAE using 40 mL of acetone at 120 °C for 20 min. The limits of quantitation ranged from 0.1 to 0.4 ng/g (dry weight) in 2 g of the sediment samples. Moreover, as the chlorinated alkyl phosphates present no molecular ions in EI, GC–MS with furan-CI (furan-CI) was applied to confirm their determination in complex environmental samples. The recoveries of the selected OPFRs in spiked sediment samples ranged from 62% to 106% (relative standard derivation, 1−11%). The total concentrations of the selected OPFR residues in marine and river sediments ranged from 1.0 to 12.6 ng/g.     

Influence of protein–protein interactions on bulk mass transport during ultrafiltration

Bulk mass transfer limitations can have a significant effect on the flux and selectivity during membrane ultrafiltration. Most previous studies of these phenomena have employed the simple stagnant film analysis, but this model is unable to account for the effects of solute–solute interactions on mass transport. We have developed a generalized framework for multicomponent mass transfer that includes both thermodynamic and hydrodynamic (frictional) interactions. Thermodynamic (virial) coefficients were evaluated from osmotic pressure data for albumin (BSA) and immunoglobulins (IgG), while hydrodynamic interaction parameters were determined from filtrate flux data obtained in a stirred cell using fully retentive membranes. The protein concentration profiles in the bulk solution were evaluated by numerical solution of the governing continuity equations incorporating the multicomponent diffusive flux. This model was used to analyze flux and protein transmission data obtained for the filtration of BSA and IgG mixtures through partially permeable membranes. The model accurately predicted the large reduction in flux and BSA transmission upon addition of IgG. These effects were due to the coupling between BSA and IgG mass transfer caused by protein–protein interactions.     

Widespread arginine phosphorylation in human cells——a novel protein PTM revealed by mass spectrometry

Arginine phosphorylation(p Arg) is recently discovered as a ubiquitous protein N-phosphorylation in bacteria.However,its prevalence and roles in mammalian cells remain largely unknown due to the lack of established workflow and the inherent lability of phosphoramidate(P–N) bond.Emerging evidences suggest that N-phosphorylation may extensively exist in eukaryotes and play crucial roles.We report a phosphoproteomic workflow,which allows for the first time revealing the widespread occurrence of p Arg in human cells by mass spectrometry.By virtue of this approach,we identified 152 high-confidence p Arg sites derived from 118 proteins.Remarkably,the discovered p Arg phosphorylation motif and gene ontology hint a possible cellular function of arginine phosphorylation which may regulate the favorability of propeptide convertase substrate.The obtained p Arg dataset paves a way for a better understanding of the biological functions of eukaryotic p Arg in the future.     

Study of archaeological artifact by chromatography–mass spectrometry

An archaeological artifact, representing a compound piece of the Bronze Age, the outer surface of which is encrusted with seeds, has been investigated. The chemical composition of the organic binder was determined by gas chromatography and mass spectrometry; X-ray fluorescence analysis was used to determine the nature of the inorganic base of the artifact.     

Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry

A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography–tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C₁₈ stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1–112.9 % and repeatability relative standard deviations of 1.9–7.2 %. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae.

Determination of cyclopiazonic acid in food and feeds by liquid chromatography–tandem mass spectrometry

A new, fast and efficient multiple reaction monitoring (MRM) high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the determination of cyclopiazonic acid (CPA) in mixed feed, wheat, peanuts and rice is presented. The analytical methodology involves sample extraction with an alkaline methanol–water mixture, defatting with hexane and quantification using HPLC–MS/MS without further treatment of sample extracts. Reversed-phase liquid chromatography using a C18 stationary phase coupled to negative mode electrospray triple quadrupole tandem mass spectrometry was applied. The limit of detection was 5 μg/kg while the limit of quantification was 20 μg/kg in the matrices investigated. The detector response was found to be linear over the range 25–250 μg/kg in feed and 25–500 μg/kg in wheat, peanuts and rice. The mean overall recoveries (n = 18) of CPA varied from 79% to 114% in the range of concentrations studied over a period of 4 months. Mean recoveries (n = 3 or 6) of CPA in wheat, peanuts and rice varied from 70% to 111%, 77% to 116% and 69% to 92%, respectively. The method was successfully applied to the analysis of feed and rice samples artificially infected with the fungal strain Penicillium commune, where the toxin was found at different levels.     

Determination of evodiamine and rutecarpine in human serum by liquid chromatography–tandem mass spectrometry

Evodiamine and rutecarpine are two kinds of indole alkaloids contained in the fruit of Evodiae fructus, which have been shown to exhibit various bioactivities in humans. A liquid chromatography–tandem mass spectrometric method (LC–MS/MS) was developed for the determination of evodiamine and rutecarpine in human serum. The serum was extracted by solid-phase extraction (SPE) and analyzed using a C18 column and a mobile phase consisting of methanol–water (85:15) solution containing 5 mmol/L ammonium formate at a flow rate of 0.5 mL/min. The mass spectrometer was operated in positive mode, employing the extracted ion chromatogram (EIC) for detection and quantitation of evodiamine (m/z 288) and rutecarpine (m/z 304). Good linear relationships between the peak area and the concentration were obtained in the ranges of 5.2–1040 ng/mL and 10.2–1020 ng/mL, with correlation coefficients (r) of 0.999 and 0.998, for evodiamine and rutecarpine, respectively. The repeatabilities (RSD, n=6) of quantitation for evodiamine and rutecarpine were 2.18–4.00% and 2.99–5.67%, respectively, and the recovery ranged from 90.5% to 98.1%. A comparative study of the different ionization and quantitation modes, including ESI–MS, ESI–MS/MS, APCI–MS and APCI–MS/MS, was also accomplished. The MS/MS fragmentation mechanism of the base peak ([M+H]⁺, m/z 304) of evodiamine was investigated in order to identify the analytes in more complicated body fluid samples.      

Characterization and quantification of chemical constituents in Fuzhuan brick tea using ultra-high-performance liquid chromatography–mass spectrometry

Fuzhuan brick tea, a distinctive dark tea fermented by microorganisms, is a traditional beverage in China throughout history. Recently, it has attracted considerable attention owing to its unique quality characteristics and potential health benefits. The aim of this study was to establish a method for the quality control of Fuzhuan brick tea for stable production. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was used to identify Fuzhuan brick tea, and the major components were chosen for further quantitative analysis. Subsequently, a quantification method was developed using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry, and its reliability was verified through methodological validation. Finally, a total of 30 compounds were identified, including catechins, flavonoids, alkaloids, and fatty acids. The established method was reliable for methodological validation and was applied to the quantitative analysis of Fuzhuan brick tea. This study provides a fundamental basis for the quality control and further studies on the component analysis of Fuzhuan brick tea.     

Determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons in human hair by gas chromatography–negative chemical ionization mass spectrometry

The study describes the determination of mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs), metabolites of PAHs, in human hair. Twelve selected OH-PAHs from two to four rings, generally determined in urine analysis, were investigated as markers of human exposure to PAHs. Following hydrolysis of hair specimens of 50–300 mg with 1 M NaOH, OH-PAHs were extracted using dichloromethane and submitted to an optimized derivatization with (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride. Compounds were then analyzed using gas chromatography–negative chemical ionization mass spectrometry (GC–NCIMS). The average inter-day and intra-day variability was 12% and 17%, respectively. The average recovery was 52% and the limits of detection and quantification ranged from 20 and 66 pmol/g for 1-OH-phenanthrene (i.e., 3.9 and 12.8 pg/mg) to 311 and 1030 pmol/g for 2-OH-benzo(c)phenanthrene (i.e., 75.9 and 251 pg/mg). The influence of hair washing with water as decontamination step, and enzymatic treatment (β-glucuronidase) to hydrolyze conjugated derivatives were also tested. The application of the developed method to the analysis of 30 hair specimens (17 from non-smoker and 13 from smoker volunteers) demonstrated inter-individual qualitative and quantitative variations. According to the easiness of hair sampling and based on the extended detection windows provided by hair analysis, this method is proposed as a new promising tool for the assessment of human chronic exposure to PAHs.     

Comparison of negative chemical ionization and electron impact ionization in gas chromatography–mass spectrometry of endocrine disrupting pesticides

The study of pesticide residues belonging to endocrine disrupting chemicals (EDCs) (23 analytes of different chemical classes – organochlorines, organophosphates, pyrethroids, dicarboximides, phtalamides, dinitroanilines, pyrazole, triazinone) in apple matrix with conventional capillary GC–NCI-MS (with methane as reagent gas) in comparison to EI ionization is presented. For sample preparation QuEChERS method was applied. The lowest calibration levels (LCLs) for all pesticides were determined in both modes. Calibration in the NCI mode was performed at the concentration levels from 0.1 to 500 μg kg⁻¹ (R² > 0.999) and for EI in the range from 5 to 500 μg kg⁻¹ (R² > 0.99). From LCLs the instrumental limits of detection (LODs) and quantification (LOQs) were calculated. Chemometric study of pesticide signals in two MS modes was performed. Repeatability of all measurements, expressed by the relative standard deviations of absolute peak areas was better than 10% for the majority of compounds. Significantly lower values were obtained for the NCI mode.     

Sampling of benzene in environmental and exhaled air by solid-phase microextraction and analysis by gas chromatography–mass spectrometry

Benzene is classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC). The risk assessment for benzene can be performed by monitoring environmental and occupational air, as well as biological monitoring through biomarkers. The present work developed and validated methods for benzene analysis by GC/MS using SPME as the sampling technique for ambient air and breath. The results of the analysis of air in parks and avenues demonstrated a significant difference, with average values of 4.05 and 18.26 μg m⁻³, respectively, for benzene. Sampling of air in the occupational environment furnished an average of 3.41 and 39.81 μg m⁻³. Moreover, the correlations between ambient air and expired air showed a significant tendency to linearity (R ² = 0.850 and R ² = 0.879). The results obtained for two groups of employees (31.91 and 72.62 μg m⁻³) presented the same trend as that from the analysis of environmental air.     

Identification and determination of mycotoxins and food additives in feed by HPLC–high-resolution time-of-flight mass spectrometry

High-resolution time-of-flight mass spectrometry combined with high performance liquid chromatography is proposed for the detection and determination of 25 mycotoxins and 8 food additives (coccidiostats) in animal feed, using simplified and rapid sample preparation. We developed a procedure for the identification and determination of analytes by the standard addition method. The lower limit of the analytical range is 1 (400) µg/kg for mycotoxins; the analytical range for coccidiostats in feed is 10–200 mg/kg. The relative standard deviation of the results does not exceed 10%. The analysis time is 0.5–1 h.